Regulation of malate metabolism in yeast
Van Staden, J
Based upon their ability to utilise dicarboxylic acids, yeast can be classified as either K+ (capable of using one or more Krebs cycle intermediate as sole carbon and energy source) or K- (unable to do so). We are investigating the transport and intracellular degradation of L-malate in the K- yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as in the K+ yeast Candida utilise. Cloning, molecular analysis and transcriptional regulation of the relevant genes will help us to better understand the regulatory mechanisms involved in the differential utilisation of L-malate and its physiological relevance. Furthermore, cloning of the different transporter and/or malic enzymes from these species will provide us with more options for heterologous expression of the appropriate genes for specific commercial applications.
Although the malic enzyme is highly conserved in prokaryotic and eukaryotic organisms, its metabolic regulation, activity and function differ between organisms and subcellular locations. Cells of S. pombe effectively convert malate to pyruvate and carbon dioxide via a cytosolic NAD-dependent malic enzyme with a high substrate affinity (Km = 3.2 mm). In contrast, S. cerevisiae has a mitochondrial NADP-dependent malic enzyme with a very low substrate affinity (Km = 50 mm), resulting in poor degradation of malate. The malic enzyme genes from both S. pombe and S. cerevisiae were cloned and further analysed to determine their regulation and metabolic function. We found that expression of the S. pombe malic enzyme gene, mae2, increased 3- and 5-fold under anaerobic conditions and when grown on 30% glucose, respectively. Several putative regulatory elements were identified in the mae2 promoter and further analyses of these elements were done to determine their role in the induced expression under fermentative conditions. Similarly, the promoter region of the S. cerevisiae malic enzyme gene, MAE1, was analysed to identify putative regulatory elements that may explain differences between wine yeast strains with regard to the degradation of malate during vinification.
Cloning and analysis of the gene encoding the S. pombe malate transporter (mae1) showed that it is responsible for the active transport of malate, but not fumarate. In contrast, cells of C. utilise actively transport both malate and fumarate, which is subject to catabolite repression and substrate induction. To gain a better understanding of the different mechanisms involved in the regulation of dicarboxylic acid metabolism in C. utilis, an in depth study of the transport protein(s) and intracellular enzymes is required. A cDNA library of C. utilis was constructed to clone the malate/fumarate transporter genes of C. utilis. The library was transformed into a S. cerevisiae strain that already carries the S. pombe malic enzyme gene, followed by screening for transformants that are able to degrade extracellular l-malate. Future research will involve the characterisation of the dicarboxylic acid transporter from C. utilis and comparison with the S. pombe malate transporter gene, mae1.
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